皮肤角朊细胞与表皮干细胞双向电泳图谱的建立及差异分析

目的 建立皮肤角朊细胞与表皮干细胞的双向电泳图谱,并分析二者表达蛋白质的差异,为进一步研究体外调控表皮十细胞的增殖、分化提供线索.方法酶消化法获取单个表皮细胞悬液.Ⅳ型胶原快速贴壁法分选表皮干细胞.利用舣向电泳技术(2-DE)建立两种细胞的蛋白质表达图谱,并用Imagemaster 2D elite 5.0软件分析两种细胞的差异表达蛋一点.结果两种细胞的2-DE蛋白表达谱具有良好的重复性和可比性.皮肤角朊细胞与表皮干细胞的电泳图谱平均蛋白质点数分别为(982 ±18)个和(930 ±15)个,匹配点数为(850±13)个和(798±11)个,匹配率是86.56%和85.81%;两种细胞蛋白质间匹配点数是(886 ±8)个,匹配率是76.98%.找到差异蛋白点11个,其中只在表皮下细胞中表达或高表达的有8个;只在皮肤角朊细胞中表达或高表达的有3个.结论利用双向电泳法得到了分辨率较高儿重复性好的皮肤角朊细胞与表皮干细胞蛋白质组图谱,且二者存在有一定的差异.     

烧伤合并神经损伤早期整形临床观察

目的：观察早期整形手术对烧伤合并神经损伤患者的临床效果。方法将2013年2月－2014年3月医院收治的40例深Ⅱ度以上烧伤合并神经损伤的患者按照治疗方法分为观察组和对照组各20例。比较2组临床疗效、感觉与运动功能恢复以及不良反应、并发症等。结果术后对2组患者进行6个月的随访,观察组神经功能总有效率为95．0％明显高于对照组的80．0％,差异有统计学意义（P＜0．05）。2组治疗后休克情况、烧伤面积较治疗前明显改善,差异有统计学意义（P＜0．05）;观察组治疗后烧伤面积较对照组改善明显,差异有统计学意义（P＜0．05）。2组均未发生严重并发症与不良反应（P＞0．05）。结论烧伤合并神经损伤患者行早期整形手术效果明显,患者感觉和运动功能恢复情况优于晚期整形手术,值得临床推广。     

角质形成细胞与表皮干细胞双向电泳图谱差异分析

1.1 主要试剂与仪器 1.1 主要试剂与仪器 pH固相梯度干胶条(IPGstrip pH 3～10,长17 cm)购自广州威佳公司,dispase Ⅱ、表皮干细胞(ESC)培养基、FBS和DMEM培养基购自美国Gibco公司,角蛋白19购自美国San-ta Cruz公司.等电聚焦仪、垂直电泳系统、透射扫描仪(Im-age Scanner Ⅱ型)及扫描软件Lab Scan均购自美国通用公司,图像分析软件Image Master 2D Elite 5.0由美Amer-sham Biosciences公司提供,FACS Calibur型流式细胞仪购自美国BD公司.     

IL-6、IL-10、PLA2在重度烧伤并发脓毒症中的变化及其预后

目的：探讨IL-6、IL-10、PLA2在重度烧伤并发脓毒症中的变化及其对预后的影响。方法选取2012年2月至2014年6月期间郑州市第一人民医院收治的重度烧伤患者100例,其中30例未合并脓毒血症（A组）,70例合并脓毒血症,52例未并发MODS为预后良好组（B组）,18例并发MODS为预后不良组（C组）。检测受试者不同时间点血清IL-6、IL-10、PLA2含量。结果3组IL-6水平均在烧伤后7 d出现明显变化（P<0.05）,烧伤后7 d,B、C组IL-6水平明显高于A组（P<0.05）,且C组IL-6水平高于B组（P<0.05）。A组IL-6水平在烧伤后7 d开始下降（P<0.05）,B组IL-6水平在烧伤后14 d开始明显下降（P<0.05）,C组IL-6水平则一直处于上升趋势（P<0.05）。3组IL-10水平均在烧伤后7 d出现明显变化（P<0.05）,烧伤后7 d,B、C组IL-10水平明显高于A组（P<0.05）,且C组IL-10水平高于B组（P<0.05）。A组IL-10水平在烧伤后7 d开始下降（P<0.05）,B组IL-10水平在烧伤后14 d开始明显下降（P<0.05）,C组IL-10水平则一直处于上升趋势（P<0.05）。3组IL-10水平均在烧伤后3 d出现明显变化（P<0.05）,烧伤7 d后变化幅度加大（P<0.05）,烧伤后7 d,B、C组PLA2水平明显高于A组（P<0.05）,且C组PLA2水平高于B组（P<0.05）。A组PLA2水平在烧伤后7 d开始下降（P<0.05）,B、C组PLA2水平在烧伤后14 d开始明显下降（P<0.05）,C组IL-10水平则一直处于上升趋势（P<0.05）。结论 IL-6、IL-10、PLA2等指标与重度烧伤并发脓毒症的发生及患者预后密切相关,因此宜对重度烧伤患者相关指标进行动态监测,以及时判断患者病情及预后。     

几丁质膜作为表皮干细胞培养支架的实验研究

Objective To investigate the feasibility of constructing a skin tissue engineering cover-ing on chitinous membrane using rat epidermal stem cells (ESCs). Methods Rat ESCs were isolated and cultured by cold digestive method and collagen type Ⅳ adherent method. Cell colonies were observed with in-verted microscope. Expressions of DNA and RNA of ESCs were detected with laser scanning confocal micro-scope. Growth curves of cells were determined with Alamar BlueTM colorimetric method. Expressions of sur-face markers of ESCs (CD29, CD71, CD49d, and CD34) were detected with flow cytometer. Positive expres-sions of CK15, CK19, and P63 of ESCs were determined by immunohistochemistry. Influence of original chi-tinous membrane leachate in different dilutions on ESCs was observed. Condition of growth of ESCs on the ve-hicle was observed. Results Isolated cultured cells were verified as ESCs, of which the doubling generation time was 48 hs. CD29 and CD49d were positive;CD71 and CD34 were negative;CKi9, CK15, and P63 were positive. Compared with that of control group, ESCs cultured in chitinous membrane leachate showed slight cell proliferation when diluted to 1:8-1:512 dilutions, but there was no statistically significant difference (P>0.05). The checkerboard-form cell colonies of ESCs could be visualized with naked eyes on the chi-tinous membrane in 2-4 weeks of culture. A multitude of ESCs were seen to grow on fibres under microscope. Conclusions Chitinous membrane may be used as ESCs culture vehicle, and biological compatibility is good.     

几丁质膜作为表皮干细胞培养支架的实验研究

Objective To investigate the feasibility of constructing a skin tissue engineering cover-ing on chitinous membrane using rat epidermal stem cells (ESCs). Methods Rat ESCs were isolated and cultured by cold digestive method and collagen type Ⅳ adherent method. Cell colonies were observed with in-verted microscope. Expressions of DNA and RNA of ESCs were detected with laser scanning confocal micro-scope. Growth curves of cells were determined with Alamar BlueTM colorimetric method. Expressions of sur-face markers of ESCs (CD29, CD71, CD49d, and CD34) were detected with flow cytometer. Positive expres-sions of CK15, CK19, and P63 of ESCs were determined by immunohistochemistry. Influence of original chi-tinous membrane leachate in different dilutions on ESCs was observed. Condition of growth of ESCs on the ve-hicle was observed. Results Isolated cultured cells were verified as ESCs, of which the doubling generation time was 48 hs. CD29 and CD49d were positive;CD71 and CD34 were negative;CKi9, CK15, and P63 were positive. Compared with that of control group, ESCs cultured in chitinous membrane leachate showed slight cell proliferation when diluted to 1:8-1:512 dilutions, but there was no statistically significant difference (P>0.05). The checkerboard-form cell colonies of ESCs could be visualized with naked eyes on the chi-tinous membrane in 2-4 weeks of culture. A multitude of ESCs were seen to grow on fibres under microscope. Conclusions Chitinous membrane may be used as ESCs culture vehicle, and biological compatibility is good.     

氧化亚氮/氧气混合气体吸入镇痛在烧伤病人中的效果观察

目的观察氧化亚氮 /氧气(N2O/O₂)混合气体吸入镇痛在烧伤病人中的应用效果。方法选取 2013年 2月至 2015年 2月郑州市第一人民医院收治的烧伤病人 120例,采用双盲法随机分为观察组(创面换药时予以 N2O/O₂混合气体吸入镇痛, n＝ 60)、对照组(创面换药时吸入氧气, n＝60)其余治疗相同,以心电监护设备评估其在吸入 N2O/O₂前(T1)、吸入 2 min(T2)、吸入 5 min(T3)、吸入 10 min(T4)的生命体征,比,较两组换药前、换药中、换药后 10 min的疼痛度［疼痛视觉模拟评分(VAS)］、焦虑程度［汉化版烧伤专用疼痛 ?焦虑量表(C?BSPAS)］、镇静程度(Ramsay镇静评分)、镇痛满意度,并记录其不良反应。结果观察组在 T2、T3、T4时心率(HR)［T2(92.33±9.31)次 /分比(96.16±9.53)次 /分、 T3(94.10±9.53)次 /分比(98.78±9.76)次 /分、 T4(91.63±9.25)次 /分比(97.45±9.88)次 /分］、收缩压(SBP)［T2(124.45±12.67)mmHg比(129.35±13.40)mmHg、T3(127.79±12.55) mmHg比(133.47±13.68)mmHg、T4(124.13±12.68)mmHg比(129.55±12.67)mmHg］低于对照组,而血氧饱和度(SpO₂)［T2(99.64±0.03)%比(95.84±0.39)%、T3(99.02±0.05)%比(97.25±0.34)%、T4(99.03±0.06)%比(96.88±0.45)%］高于对照组,观察组 T2时舒张压(DBP)低于对照组［(71.38±7.26)mmHg比(74.58±7.62)mmHg,P＜0.05］;观察组换药中、换药后 10 min VAS、C? BSPAS评分低于对照组(P＜0.05);观察组镇静分级优于对照组(P＜0.05);观察组镇痛满意度 93.33%高于对照组 80.00%(P＜ 0.05);两组不良反应发生率比较差异无统计学意义(P＞0.05)。结论 N2O/O₂混合气体吸入镇痛应用于烧伤病人中有较好的镇痛、镇静效果,同时降低生命体征波动及焦虑程度,提高病人镇痛满意度,安全可靠,值得在临床推广应用。     

不同培养体系对鼠表皮干细胞增殖和分化的影响

目的 探讨不同培养体系对表皮干细胞增殖、分化的影响,建立理想的调控表皮干细胞增殖、分化的培养体系.方法 酶消化和Ⅳ型胶原快速黏附法获取鼠表皮干细胞.分别在普通培养皿培养、与几丁质膜生物支架材料共培养及以几丁质膜生物支架材料作为载体植入裸鼠体内培养等不同培养体系下观察表皮干细胞生长情况.普通培养和与几丁质膜生物支架材料共培养4周后,对比表皮干细胞克隆形成率的差异.免疫组织化学染色观察表皮干细胞以几丁质膜为载体植入裸鼠体内后4周表皮干细胞的增殖、分化情况.结果 表皮干细胞在普通培养皿培养3d左右,细胞开始克隆增殖;12 d左右融合成片;传代培养后增殖能力逐渐减低,融合成片时间逐渐延长,传代培养3～4代后细胞终末分化,失去增殖能力.几丁质膜生物支架材料培养表皮干细胞,2周后呈棋盘式集落生长,几丁质膜生物支架材料上有大量的表皮干细胞小集落,集落上有大量的增殖细胞附着生长,扫描电镜下见几丁质膜生物支架材料纤维直径约10 μm,以纤维为主,上下两层呈纵横排列成十字孔,孔间有大量表皮干细胞集落.几丁质膜生物支架材料培养表皮干细胞4周后,其克隆形成率明显高于普通培养皿培养[(12.6±2.7)％比(5.7±1.1)％,P＜0.05].表皮干细胞几丁质膜生物支架材料植入裸鼠体内培养4周后,细胞大量增殖形成巢状排列,在表皮干细胞巢周围,可见有类似皮肤附件结构.结论 表皮干细胞在体外普通培养皿培养可增殖生长,但维持增殖时间较短;与几丁质膜生物支架材料共培养,可较长时间地维持表皮干细胞的增殖特性;植入体内后表皮干细胞大量增殖.     

几丁质膜作为表皮干细胞培养支架的实验研究

Objective To investigate the feasibility of constructing a skin tissue engineering cover-ing on chitinous membrane using rat epidermal stem cells (ESCs). Methods Rat ESCs were isolated and cultured by cold digestive method and collagen type Ⅳ adherent method. Cell colonies were observed with in-verted microscope. Expressions of DNA and RNA of ESCs were detected with laser scanning confocal micro-scope. Growth curves of cells were determined with Alamar BlueTM colorimetric method. Expressions of sur-face markers of ESCs (CD29, CD71, CD49d, and CD34) were detected with flow cytometer. Positive expres-sions of CK15, CK19, and P63 of ESCs were determined by immunohistochemistry. Influence of original chi-tinous membrane leachate in different dilutions on ESCs was observed. Condition of growth of ESCs on the ve-hicle was observed. Results Isolated cultured cells were verified as ESCs, of which the doubling generation time was 48 hs. CD29 and CD49d were positive;CD71 and CD34 were negative;CKi9, CK15, and P63 were positive. Compared with that of control group, ESCs cultured in chitinous membrane leachate showed slight cell proliferation when diluted to 1:8-1:512 dilutions, but there was no statistically significant difference (P>0.05). The checkerboard-form cell colonies of ESCs could be visualized with naked eyes on the chi-tinous membrane in 2-4 weeks of culture. A multitude of ESCs were seen to grow on fibres under microscope. Conclusions Chitinous membrane may be used as ESCs culture vehicle, and biological compatibility is good.     

依达拉奉对脂多糖引起的小鼠急性肺损伤的保护效应研究

[目的]探讨依达拉奉在急性肺损伤当中的保护效应及其可能的机制。[方法]成年C57小鼠54只被随机等分为3组:正常组(假手术组),对照组(LPS+生理盐水)和实验组(LPS+依达拉奉)。将对照组和实验组小鼠,采用气道内注入LPS溶液(2 mg/mL)1 mL/kg造急性肺损伤模型,正常组小鼠不注。实验组小鼠尾静脉注射依达拉奉溶液(2 mg/mL)2.5 mL/kg,对照组小鼠注射等体积生理盐水。24 h后,每组取6只小鼠腹腔注射过量麻醉戊巴比妥(100 mg/kg),后断头处死,取肺组织用于组织学及丙二醛(MDA)、超氧化物歧化酶(SOD)、TNF-α,IL-1β和IL-6的检测。每组余下的12只小鼠用做生存率实验,每12 h观测1次并记录小鼠存活的情况,观察周期为4d。[结果]实验组小鼠生存率明显高于对照组(P<0.05);LPS刺激后,小鼠肺组织内的MDA含量由(1.41±0.119)nmol/mg上升到(4.48±0.159)nmol/mg,而依达拉奉治疗后则降到(2.56±0.157)nmol/mg(P<0.01);LPS刺激后,小鼠肺组织内的SOD活力由(12.86±1.49)U/mg下降到(8.95±1.02)U/mg,而依达拉奉治疗后则上升至(10.69±1.77)U/mg(P<0.01);同时,LPS刺激后,小鼠肺组织内的TNF-α,IL-1β和IL-6含量分别由(47.89±4.71)pg/mg、(79.39±3.45)pg/mg和(81.9±6.39)pg/mg上升到(300.48±12.18)pg/mg、(717.99±35.01)pg/mg和(428.99±21.89)pg/mg,而依达拉奉治疗后小鼠肺组织的含量则降到(191.84±6.43)pg/mg、(236.87±13.46)pg/mg和(136.92±12.47)pg/mg。病理结果显示:依达拉奉减轻了LPS引起的肺水肿,弥漫性出血和炎性细胞的浸润等症状。[结论]依达拉奉可以减轻LPS引起的急性肺损伤。